Infections by Pseudomonas aeruginosa and Corynebacterium diphtheria are still very common worldwide. Diphtheria infections are still endemic in several regions, including Africa, India, Bangladesh, Vietnam, South America and Russia, and cases are reported all over the world. Recent outbreaks of diphtheria have also been reported in countries like the Newly Independent States of the former Soviet Union, and in poor, socio-economically disadvantaged groups living in crowded conditions of Europe and the United States. Diphtheria infections are still the cause of numbers of deaths in the world, and are still important despite the vast vaccination programs against this pathogen. The re-emergence of epidemic in countries where vaccinations and immunizations have been performed could be explained by the introduction of a new biotype of toxigenic C. diphtheria and a large gap of immunity among adults. In fact, it is well known that the level of immunity declines in late childhood and adolescence, and some serological surveys demonstrated that 20% to >50% of adolescents and adults lack immunity to diphtheria toxin in the US. Even when properly treated, between 5%-10% of diphtheria patients will die from this infection.
P. aeruginosa is a Gram-negative bacillus ubiquitously present in the environment and, according to the Centers for Disease Control and Prevention, the fourth most commonly isolated nosocomial pathogen. Nearly all P. aeruginosa clinical cases are associated with compromised host defense. Systemic infections are also common in patients with severe burns, and in immunosuppressed AIDS and cancer patients. The infection by P. aeruginosa can also be seen with contact lenses wearers that develop keratitis of the cornea. In addition, P. aeruginosa is responsible for ventilator-acquired pneumonia, and it is the primary cause of mortality in cystic fibrosis patients due to lung infection.
It is well known that the diphtheria toxin (DT) is the virulence factor of C. diphtheria, and that exotoxin A (ETA) is one of the many virulence factors of P. aeruginosa, and it has been shown to be produced by 95% of P. aeruginosa clinical isolates. It has been reported that ETA deficient strains are twenty times less virulent in mice than wild-type strains (Miyazaki, S. et al., J Med Microbiol 43:169-75, 1995), and that immunization directed against ETA increased survival in normal and thermally injured mice infected by P. aeruginosa (El-Zaim, H. S. et al., Infect Immun 66:5551-4, 1998). For DT, vaccination with a non-toxic mutant DT is widely used worldwide to prevent diphtheria infections.
The diphtheria toxin (DT) and the Pseudomonas exotoxin A (ETA) are two bacterial toxins having A and B subunits. These toxins are characterized by a B moiety that recognizes the cell surface receptor but that also plays a role in the translocation of the toxin into the cytosol, and an A moiety that contains the catalytic activity of the toxin. When released into the cytosol, the A subunit can inactivate the elongation factor-2 (EF-2) by inducing the ADP-ribosylation of a modified histidine residue called diphthamide, thus leading to cell death by blocking protein translation. The A subunit of DT and ETA present a high homology with the A subunit of other ADP-rybosyltransferases such as the cholera toxin of Vibrio cholerae, the heat-labile enterotoxin from Escherichia coli, the pertussis toxin from Bordetella pertussis, the C3-like exoenzyme from Clostridium botulinum and Clostridium limosum, as well as putative ADP-rybosyltransferases from Neisseria gonorrhoeae, Staphylococcus aureus and Thermoanaerobacter teng. 
Diphthamide biosynthesis occurs on His715 of EF-2 (His699 in yeast) following the translation of EF-2, and consists of stepwise additions on its side chain (Liu, S. et al., Mol Cell Biol 24:9487-97, 2004). Five proteins have been identified as being involved in this process in yeast and mammals, namely Dph1 to Dph5 (Liu, S. et al., 2004). The biosynthesis of diphthamide occurs in three successive steps, involving Dph1, Dph2, Dph3 and Dph4 in the first step (3-amino-3-carboxypropyl transfer), and Dph5 in the second step (methyl transfer) (Liu, S. et al., 2004). So far, no protein has been identified as participating in the third step. This is at least partly due to the fact that the intermediate product diphtine resulting of the completion of the second step can also be ADP-ribosylated.
The biological role of diphthamide has not yet been determined, but it is found in all eukaryotic organisms and in archaebacteria except eubacteria, suggesting a relevant role in cell physiology (Liu, S. et al., Mol Cell Biol 26:3835-41, 2006). In the current state of the art, EF-2 is the only protein known with certainty to contain a diphthamide residue. But even if the role of diphthamide is unclear, some reports tentatively claim that it may play a role in EF-2 regulation, structure or stability (Kimata, Y., and Kohno, K., J Biol Chem 269:13497-501, 1994; Ortiz, P. A., and Kinzy, T. G., Nucleic Acids Res 33:5740-8, 2005).
Dph proteins are encoded by dph genes, which are highly evolutionary conserved among eukaryotes, thus suggesting that they have an important role in cell biology. Several yeasts and CHO cell lines lacking the expression of the different dph genes have been generated after exposure to mutagens (Chen, J. et al., Mol Cell Biol 5:3357-60, 1985; Kohno, K., T. et al., Somat Cell Mol Genet. 11:421-31, 1985), but none of these show distinctive phenotypes other than DT and ETA resistance, except for dph3.
The dph3 gene has been shown to be essential during mouse development (Liu, S. et al., 2006), since the loss of both dph3 alleles is lethal for the embryo. Furthermore, Saccharomyces cerevisiae cells lacking the capacity to express dph3 gene present growth defects and increased sensitivity to temperature and drugs (Liu, S. et al., 2004). Dph3 protein has been further shown to physically interact with the elongator complex in yeast, and its absence in yeast cell lines leads to the inhibition of the toxic action of zymocin (Fichtner, L. Et al., Mol Microbiol 49:1297-307, 2003). Dph3 seems to prevent the proteolysis of a protein that is part of the elongator complex, thus suggesting an important role of Dph3 in this complex's function/regulation. Dph1 and Dph2 proteins have also been shown to interact with the elongator complex by TAP-tagging. Therefore, those three proteins seem to play a certain role in other processes than just the biosynthesis of diphthamide.
More recently, it has been shown that the elongator complex and Dph3 are both required in yeast for the biosynthesis of modified nucleosides present at the wobble position in tRNA (Huang, B. et al., RNA 11:424-36, 2005). These modified nucleosides seem to be mostly involved in the decoding process of mRNA, in addition to acting as identity elements in amino-acyl-tRNA synthetase recognition.
The dph1 gene has also been cloned independently as ovca1 in ovarian cancer cells (Chen, C. M., and Behringer, R. R., Genes Dev 18:320-32, 2004) where its expression is absent in about 80% of the tumors. In mice, dph1 acts as a tumor suppressor, as knockout mouse embryonic fibroblasts (MEFs) show proliferation defects related to a reduction of retinoblastoma 1 (Rb1) phosphorylation. It has also been shown by the same group that the loss of tumor protein 53 (p53) conferred the ability to rescue the proliferation defects of ovca1-knockout MEFs. Foremost, the ovca1 heterozygote mice develop cancer spontaneously. The dph1 gene is also essential to mouse development as the ovca1−/− mice die at birth or before (Chen, C. M., and Behringer, R. R., 2004).
In the 1940s and 1950s, a vaccine program based on diphtheria toxoid had nearly eliminated diphtheria in industrialized countries. However, recent outbreaks of diphtheria have been reported in various countries including Russia and the newly independent states of the former Soviet Union, and in socio-economically disadvantaged groups living in crowded conditions in Europe and in the US. The level of immunity against DT declines in late childhood and adolescence, and serological surveys showed that more than 50% of adults lack immunity to DT in some industrialized countries. Therefore, the lack of immunity against diphtheria in adults represents a potential threat that could lead to the development of epidemics in industrialized countries. Since those pathogens are still a major cause of many health problems, some resulting in death, and despite immunization programs and the use of antibiotics, new ways of treating/preventing infections caused by both C. diphtheria and P. aeruginosa are highly desirable.
Autologous stem cell transplantation (ASCT) can be used for patients with hematologic malignancies like Hodgkin and non-Hodgkin lymphomas, multiple myelomas and leukemias, as well as for other tumor types. High-dose chemotherapy, total body irradiation or salvage therapy are often used as frontline treatments of these malignancies, thus making ASCT needed for stem cell support. Unfortunately, minimal residual disease is often present in bone marrow or peripheral blood of cancer patients, and purging techniques have yet to be developed in order to eliminate those contaminating tumor cells. Such purging techniques can be performed ex vivo with chemotherapeutic drugs (such as cyclophosphamide-derived drugs), monoclonal antibodies and complement, and negative or positive selection (CD34+). In vivo purging is also an option with the use of Rituximab (an anti-CD20 monoclonal antibody) and chemotherapeutic agents for B cell malignancies. However, several side effects are associated with those ex vivo or in vivo purging approaches, such as delayed engraftment, loss of progenitor cells and high frequency of life-threatening infections. Furthermore, these techniques also appears to have an insufficient purging efficacy. US patent 2005/0287116 teaches the transfection of a mutant hamster EF-2 gene into cells, the mutant hamster EF-2 presenting a Arg717 mutation changing the arginine for a glycine. This was shown to confer sufficient cell resistance to diphtheria toxin for the production of adenoviral vectors having the capability of carrying DT subunit DT-A.
It would also be highly desirable to have a resistance gene, that is not an antibiotic resistance gene, and that can be used as a genetic marker to select for genetically modified cells.